Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that processFil: Salinas Ojeda, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ortiz Flores, Rodolfo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Distel, Jesús Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Aguilera, Milton Osmar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Beron, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Anhydrobiosis and Freezing-Tolerance:Adaptations That Facilitate the Establishment of Panagrolaimus Nematodes in Polar Habitats

<div><p>Anhydrobiotic animals can survive the loss of both free and bound water from their cells. While in this state they are also resistant to freezing. This physiology adapts anhydrobiotes to harsh environments and it aids their dispersal. <i>Panagrolaimus davidi</i>, a bacterial feeding anhydrobiotic nematode isolated from Ross Island Antarctica, can survive intracellular ice formation when fully hydrated. A capacity to survive freezing while fully hydrated has also been observed in some other Antarctic nematodes. We experimentally determined the anhydrobiotic and freezing-tolerance phenotypes of 24 <i>Panagrolaimus</i> strains from tropical, temperate, continental and polar habitats and we analysed their phylogenetic relationships. We found that several other <i>Panagrolaimus</i> isolates can also survive freezing when fully hydrated and that tissue extracts from these freezing-tolerant nematodes can inhibit the growth of ice crystals. We show that <i>P. davidi</i> belongs to a clade of anhydrobiotic and freezing-tolerant panagrolaimids containing strains from temperate and continental regions and that <i>P. superbus</i>, an early colonizer at Surtsey island, Iceland after its volcanic formation, is closely related to a species from Pennsylvania, USA. Ancestral state reconstructions show that anhydrobiosis evolved deep in the phylogeny of <i>Panagrolaimus</i>. The early-diverging <i>Panagrolaimus</i> lineages are strongly anhydrobiotic but weakly freezing-tolerant, suggesting that freezing tolerance is most likely a derived trait. The common ancestors of the <i>davidi</i> and the <i>superbus</i> clades were anhydrobiotic and also possessed robust freezing tolerance, along with a capacity to inhibit the growth and recrystallization of ice crystals. Unlike other endemic Antarctic nematodes, the life history traits of <i>P. davidi</i> do not show evidence of an evolved response to polar conditions. Thus we suggest that the colonization of Antarctica by <i>P. davidi</i> and of Surtsey by <i>P. superbus</i> may be examples of recent “ecological fitting” of freezing-tolerant anhydrobiotic propagules to the respective abiotic conditions in Ross Island and Surtsey.</p></div

Expression of Ixodes scapularis Antifreeze Glycoprotein Enhances Cold Tolerance in Drosophila melanogaster

Drosophila melanogaster experience cold shock injury and die when exposed to low non-freezing temperatures. In this study, we generated transgenic D. melanogaster that express putative Ixodes scapularis antifreeze glycoprotein (IAFGP) and show that the presence of IAFGP increases the ability of flies to survive in the cold. Male and female adult iafgp-expressing D. melanogaster exhibited higher survival rates compared with controls when placed at non-freezing temperatures. Increased hatching rates were evident in embryos expressing IAFGP when exposed to the cold. The TUNEL assay showed that flight muscles from iafgp-expressing female adult flies exhibited less apoptotic damage upon exposure to non-freezing temperatures in comparison to control flies. Collectively, these data suggest that expression of iafgp increases cold tolerance in flies by preventing apoptosis. This study defines a molecular basis for the role of an antifreeze protein in cryoprotection of flies

Stress-induced c-Fos expression is differentially modulated by dexamethasone, diazepam and imipramine

Immobilization stress upregulates c-Fos expression in several CNS areas. Repeated stress or the use of drugs can modulate stress-induced c-Fos expression. Here, we investigated in 40 different areas of the rat brain the effects of dexamethasone (SDX, a synthetic glucocorticoid), diazepam (SBDZ, a benzodiazepine), and imipramine (IMI, an antidepressant) on the c-Fos expression induced by restraint stress. Wistar rats were divided into four groups and submitted to 20 days of daily injection of saline (three first groups) or imipramine, 15 mg/kg, i.p. On day 21, animals were submitted to injections of saline (somatosensory, SS), SDX (1 mg/kg, i.p.), SBDZ (5 mg/kg, i.p.), or IMI (15 mg/kg, i.p.) before being submitted to restraint. Immediately after stress, the animals were perfused and their brains processed with immunohistochemistry for c-Fos (Ab-5 Oncogene Science). Dexamethasone reduced stress- induced c-Fos expression in SS cortex, hippocampus, paraventricular nucleus of the hypothalamus (PVH), and locus coeruleus (LC), whereas diazepam reduced c-Fos staining in the SS cortex, hippocampus, bed nucleus of stria terminalis, septal area, and hypothalamus (preoptic area and supramammillary nucleus). Chronic administration of imipramine decreased staining in the hippocampus, PVH, and LC, while increasing it in the nucleus raphe pallidus. We conclude that dexamethasone, diazepam and imipramine differentially modulate stress-induced Fos expression. the present study provides an important comparative background that may help in the further understanding of the effects of these compounds and on the brain activation as well as on the behavioral, neuroendocrine, and autonomic responses to stress.UFRRJ, Dept Physiol Sci, BR-23890000 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psychobiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psychobiol, São Paulo, BrazilWeb of Scienc

Long-Term Cold Acclimation Extends Survival Time at 0°C and Modifies the Metabolomic Profiles of the Larvae of the Fruit Fly Drosophila melanogaster

Drosophila melanogaster is a chill-susceptible insect. Previous studies on this fly focused on acute direct chilling injury during cold shock and showed that lower lethal temperature (LLT, approximately -5°C) exhibits relatively low plasticity and that acclimations, both rapid cold hardening (RCH) and long-term cold acclimation, shift the LLT by only a few degrees at the maximum.We found that long-term cold acclimation considerably improved cold tolerance in fully grown third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt(50)) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Such marked physiological plasticity in Lt(50) (in contrast to LLT) suggested that chronic indirect chilling injury at 0°C differs from that caused by cold shock. Long-term cold acclimation modified the metabolomic profiles of the larvae. Accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM) were the two most prominent responses. In addition, restructuring of the glycerophospholipid composition of biological membranes was observed. The relative proportion of glycerophosphoethanolamines (especially those with linoleic acid at the sn-2 position) increased at the expense of glycerophosphocholines.Third-instar larvae of D. melanogaster improved their cold tolerance in response to long-term cold acclimation and showed metabolic potential for the accumulation of proline and trehalose and for membrane restructuring

Pooled extracellular receptor-ligand interaction screening using CRISPR activation.

Extracellular interactions between cell surface receptors are necessary for signaling and adhesion but identifying them remains technically challenging. We describe a cell-based genome-wide approach employing CRISPR activation to identify receptors for a defined ligand. We show receptors for high-affinity antibodies and low-affinity ligands can be unambiguously identified when used in pools or as individual binding probes. We apply this technique to identify ligands for the adhesion G-protein-coupled receptors and show that the Nogo myelin-associated inhibitory proteins are ligands for ADGRB1. This method will enable extracellular receptor-ligand identification on a genome-wide scale

Model of SNARE-Mediated Membrane Adhesion Kinetics

SNARE proteins are conserved components of the core fusion machinery driving diverse membrane adhesion and fusion processes in the cell. In many cases micron-sized membranes adhere over large areas before fusion. Reconstituted in vitro assays have helped isolate SNARE mechanisms in small membrane adhesion-fusion and are emerging as powerful tools to study large membrane systems by use of giant unilamellar vesicles (GUVs). Here we model SNARE-mediated adhesion kinetics in SNARE-reconstituted GUV-GUV or GUV-supported bilayer experiments. Adhesion involves many SNAREs whose complexation pulls apposing membranes into contact. The contact region is a tightly bound rapidly expanding patch whose growth velocity increases with SNARE density . We find three patch expansion regimes: slow, intermediate, fast. Typical experiments belong to the fast regime where depends on SNARE diffusivities and complexation binding constant. The model predicts growth velocities s. The patch may provide a close contact region where SNAREs can trigger fusion. Extending the model to a simple description of fusion, a broad distribution of fusion times is predicted. Increasing SNARE density accelerates fusion by boosting the patch growth velocity, thereby providing more complexes to participate in fusion. This quantifies the notion of SNAREs as dual adhesion-fusion agents

Destruction of Dopaminergic Neurons in the Midbrain by 6-Hydroxydopamine Decreases Hippocampal Cell Proliferation in Rats: Reversal by Fluoxetine

Background Non-motor symptoms (e.g., depression, anxiety, and cognitive deficits) in patients with Parkinson disease (PD) precede the onset of the motor symptoms. Although these symptoms do not respond to pharmacological dopamine replacement therapy, their precise pathological mechanisms are currently unclear. The present study was undertaken to examine whether the unilateral 6-hydroxydopamine (6-OHDA) lesion to the substantia nigra pars compacta (SNc), which represents a model of long-term dopaminergic neurotoxicity, could affect cell proliferation in the adult rat brain. Furthermore, we examined the effects of the selective serotonin reuptake inhibitor (SSRI) fluoxetine and the selective noradrenaline reuptake inhibitor maprotiline on the reduction in cell proliferation in the subgranular zone (SGZ) by the unilateral 6-OHDA lesion. Methodology/Principal Findings A single unilateral injection of 6-OHDA into the rat SNc resulted in an almost complete loss of tyrosine hydroxylase (TH) immunoreactivity in the striatum and SNc, as well as in reductions of TH-positive cells and fibers in the ventral tegmental area (VTA). On the other hand, an injection of vehicle alone showed no overt change in TH immunoreactivity. A unilateral 6-OHDA lesion to SNc significantly decreased cell proliferation in the SGZ ipsilateral to the 6-OHDA lesion, but not in the contralateral SGZ or the subventricular zone (SVZ), of rats. Furthermore, subchronic (14 days) administration of fluoxetine (5 mg/kg/day), but not maprotiline significantly attenuated the reduction in cell proliferation in the SGZ by unilateral 6-OHDA lesion. Conclusions/Significance The present study suggests that cell proliferation in the SGZ of the dentate gyrus might be, in part, under dopaminergic control by SNc and VTA, and that subchronic administration of fluoxetine reversed the reduction in cell proliferation in the SGZ by 6-OHDA. Therefore, SSRIs such as fluoxetine might be potential therapeutic drugs for non-motor symptoms as well as motor symptoms in patients with PD, which might be associated with the reduction in cell proliferation in the SGZ

Environmental Enrichment Induces Behavioral Recovery and Enhanced Hippocampal Cell Proliferation in an Antidepressant-Resistant Animal Model for PTSD

Background: Post traumatic stress disorder (PTSD) can be considered the result of a failure to recover after a traumatic experience. Here we studied possible protective and therapeutic aspects of environmental enrichment (with and without a running wheel) in Sprague Dawley rats exposed to an inescapable foot shock procedure (IFS). Methodology/Principal Findings: IFS induced long-lasting contextual and non-contextual anxiety, modeling some aspects of PTSD. Even 10 weeks after IFS the rats showed reduced locomotion in an open field. The antidepressants imipramine and escitalopram did not improve anxiogenic behavior following IFS. Also the histone deacetylase (HDAC) inhibitor sodium butyrate did not alleviate the IFS induced immobility. While environmental enrichment (EE) starting two weeks before IFS did not protect the animals from the behavioral effects of the shocks, exposure to EE either immediately after the shock or one week later induced complete recovery three weeks after IFS. In the next set of experiments a running wheel was added to the EE to enable voluntary exercise (EE/VE). This also led to reduced anxiety. Importantly, this behavioral recovery was not due to a loss of memory for the traumatic experience. The behavioral recovery correlated with an increase in cell proliferation in hippocampus, a decrease in the tissue levels of noradrenalin and increased turnover of 5-HT in prefrontal cortex and hippocampus. Conclusions/Significance: This animal study shows the importance of (physical) exercise in the treatment of psychiatri

Parallel molecular routes to cold adaptation in eight genera of New Zealand stick insects.

The acquisition of physiological strategies to tolerate novel thermal conditions allows organisms to exploit new environments. As a result, thermal tolerance is a key determinant of the global distribution of biodiversity, yet the constraints on its evolution are not well understood. Here we investigate parallel evolution of cold tolerance in New Zealand stick insects, an endemic radiation containing three montane-occurring species. Using a phylogeny constructed from 274 orthologous genes, we show that stick insects have independently colonized montane environments at least twice. We compare supercooling point and survival of internal ice formation among ten species from eight genera, and identify both freeze tolerance and freeze avoidance in separate montane lineages. Freeze tolerance is also verified in both lowland and montane populations of a single, geographically widespread, species. Transcriptome sequencing following cold shock identifies a set of structural cuticular genes that are both differentially regulated and under positive sequence selection in each species. However, while cuticular proteins in general are associated with cold shock across the phylogeny, the specific genes at play differ among species. Thus, while processes related to cuticular structure are consistently associated with adaptation for cold, this may not be the consequence of shared ancestral genetic constraints